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1.
Neurobiol Dis ; 143: 105011, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32653674

RESUMEN

Progressive accumulation of hyperphosphorylated tau is a hallmark of various neurodegenerative disorders including Alzheimer's disease. However, to date, the functional effects of tau pathology on brain network connectivity remain poorly understood. To directly interrogate the impact of tau pathology on functional brain connectivity, we conducted a longitudinal experiment in which we monitored a fibril-seeded hTau.P301L mouse model using correlative whole-brain microscopy and resting-state functional MRI. Despite a progressive aggravation of tau pathology across the brain, the major resting-state networks appeared unaffected up to 15 weeks after seeding. Targeted analyses also showed that the connectivity of regions with high levels of hyperphosphorylated tau was comparable to that observed in controls. In line with the ostensible retention of connectivity, no behavioural changes were detected between seeded and control hTau.P301L mice as determined by three different paradigms. Our data indicate that seeded tau pathology, with accumulation of tau aggregates throughout different regions of the brain, does not alter functional connectivity or behaviour in this mouse model. Additional correlative functional studies on different mouse models should help determine whether this is a generalizable trait of tauopathies.


Asunto(s)
Encéfalo/fisiopatología , Red Nerviosa/fisiopatología , Vías Nerviosas/fisiopatología , Agregación Patológica de Proteínas/fisiopatología , Proteínas tau/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Ratones , Red Nerviosa/patología , Vías Nerviosas/patología , Agregación Patológica de Proteínas/patología
2.
Neurobiol Dis ; 127: 398-409, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30878534

RESUMEN

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.


Asunto(s)
Encéfalo/metabolismo , Microglía/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Ratones Transgénicos , Microglía/patología , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación , Tauopatías/patología
3.
J Cell Sci ; 128(3): 541­52, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25501810

RESUMEN

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, but the precise physiological function of the protein remains ill-defined. Recently, our group proposed a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses in the Drosophila melanogaster neuromuscular junctions.Flies harbor only one Lrrk gene, which might encompass the functions of both mammalian LRRK1 and LRRK2. We therefore studied the role of LRRK2 in mammalian synaptic function and provide evidence that knockout or pharmacological inhibition of LRRK2 results in defects in synaptic vesicle endocytosis, altered synaptic morphology and impairments in neurotransmission. In addition, our data indicate that mammalian endophilin A1 (EndoA1,also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73 and S75, two residues in the BAR domain. Hence, our results indicate that LRRK2 kinase activity has an important role in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequent neurotransmission at the synapse.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis/genética , Proteínas Serina-Treonina Quinasas/genética , Transmisión Sináptica/genética , Vesículas Sinápticas/genética , Animales , Células Cultivadas , Clatrina/metabolismo , Drosophila melanogaster , Dinamina I/antagonistas & inhibidores , Endocitosis/efectos de los fármacos , Hipocampo/citología , Hidrazonas/farmacología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Long-Evans , Sacarosa/farmacología , Transmisión Sináptica/efectos de los fármacos
4.
Acta Neuropathol ; 131(4): 549-69, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26739002

RESUMEN

Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aß) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer's Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aß-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aß, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aß can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aß-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aß provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aß, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aß-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in AD.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Proteínas Priónicas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Ratones Transgénicos , Mutación/genética , Presenilina-1/genética , Presenilina-1/metabolismo , Proteínas Priónicas/ultraestructura , Agregación Patológica de Proteínas/inducido químicamente , Agregación Patológica de Proteínas/patología , Tauopatías/genética , Transfección , Proteínas tau/genética , Proteínas tau/ultraestructura
5.
Neurobiol Dis ; 73: 83-95, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25220759

RESUMEN

Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.


Asunto(s)
Tauopatías , Proteínas tau/administración & dosificación , Proteínas tau/genética , Factores de Edad , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Ovillos Neurofibrilares/metabolismo , Tauopatías/inducido químicamente , Tauopatías/genética , Tauopatías/patología , Proteínas tau/química
6.
Front Aging Neurosci ; 16: 1459134, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39381137

RESUMEN

Background: Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1ß, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1ß and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. Methods: In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1ß secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in full-body Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Results: Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. Conclusion: The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors.

7.
J Alzheimers Dis ; 93(1): 151-167, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36970909

RESUMEN

BACKGROUND: Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. OBJECTIVE: We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. METHODS: Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. RESULTS: Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. CONCLUSION: The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.


Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Ratones , Humanos , Animales , Proteínas tau/metabolismo , Tauopatías/patología , Enfermedad de Alzheimer/patología , Ratones Transgénicos , Modelos Animales de Enfermedad , Anticuerpos Monoclonales , Encéfalo/patología
8.
Life (Basel) ; 13(10)2023 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-37895469

RESUMEN

BACKGROUND: The Tau58/2 and Tau58/4 mouse lines expressing 0N4R tau with a P301S mutation mimic aspects of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In a side-by-side comparison, we report the age-dependent development of cognitive, motor, and behavioral deficits in comparison with the spatial-temporal evolution of cellular tau pathology in both models. METHODS: We applied the SHIRPA primary screen and specific neuromotor, behavioral, and cognitive paradigms. The spatiotemporal development of tau pathology was investigated immunohistochemically. Levels of sarkosyl-insoluble paired helical filaments were determined via a MesoScale Discovery biomarker assay. RESULTS: Neuromotor impairments developed from age 3 months in both models. On electron microscopy, spinal cord neurofibrillary pathology was visible in mice aged 3 months; however, AT8 immunoreactivity was not yet observed in Tau58/4 mice. Behavioral abnormalities and memory deficits occurred at a later stage (>9 months) when tau pathology was fully disseminated throughout the brain. Spatiotemporally, tau pathology spread from the spinal cord via the midbrain to the frontal cortex, while the hippocampus was relatively spared, thus explaining the late onset of cognitive deficits. CONCLUSIONS: Our findings indicate the face and construct validity of both Tau58 models, which may provide new, valuable insights into the pathologic effects of tau species in vivo and may consequently facilitate the development of new therapeutic targets to delay or halt neurodegenerative processes occurring in tauopathies.

9.
Mol Ther Methods Clin Dev ; 31: 101158, 2023 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-38074413

RESUMEN

Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells.

10.
Med ; 3(12): 860-882.e15, 2022 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-36257298

RESUMEN

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.


Asunto(s)
Barrera Hematoencefálica , Transcitosis , Barrera Hematoencefálica/metabolismo , Transcitosis/fisiología , Receptores de Transferrina , Transporte Biológico/fisiología , Anticuerpos
11.
Alzheimers Dement (Amst) ; 13(1): e12204, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34095436

RESUMEN

INTRODUCTION: Diagnosis of Alzheimer's disease (AD) based on amyloid beta (A), pathologic tau (T), and neurodegeneration (N) biomarkers in peripheral fluids promises to accelerate clinical trials and intercept disease earlier. METHODS: Qualification of a Simoa plasma p217+tau assay was performed, followed by clinical utility evaluation in a cohort of 227 subjects with broad A and T spectrum. RESULTS: The p217+tau plasma assay was accurate, precise, dilution linear, and highly sensitive. All measured samples were within linear range of the assay, presented higher concentration in AD versus healthy controls (P < .0001), and plasma and cerebrospinal fluid levels correlated (r2 = 0.35). The plasma p217+tau results were predictive of central T and A status (area under the curve = 0.90 and 0.90, respectively) with low false +/- rates. DISCUSSION: The assay described here exhibits good technical performance and shows potential as a highly accurate peripheral biomarker for A or T status in AD and cognitively normal subjects.

12.
J Alzheimers Dis ; 77(4): 1417-1430, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32831201

RESUMEN

BACKGROUND: Early and accurate detection and staging is critical to managing Alzheimer's disease (AD) and supporting clinical trials. Cerebrospinal fluid (CSF) biomarkers for amyloid-ß peptides, tau species, and various neurodegenerative and inflammatory analytes are leading the way in this regard, yet there is room for improved sensitivity and specificity. In particular tau is known to be present in many different fragments, conformations, and post-translationally modified forms. While the exact tau species that might best reflect AD pathology is unknown, a growing body of evidence suggests that forms with high levels of phosphorylation in the mid-region may be especially enriched in AD. OBJECTIVE: Develop an assay for measuring p217tau in CSF. METHODS: Here we describe the development and validation of a novel sELISA for measuring CSF tau species containing phosphorylation at threonines 212 & 217, aka p217 + tau, using the PT3 antibody. RESULTS: While the analyte is present at extremely low levels the assay is sufficiently sensitive and specific to quantitate p217 + tau with excellent precision, accuracy, and dilution linearity, allowing good differentiation between diagnostic subgroups. The p217 + tau measurements appear to track AD pathology better than the commonly used p181tau epitope, suggesting superior diagnostic and staging performance. Finally, the assay can also be configured to differentiate antibody-bound versus antibody-free tau, and therefore can be used to measure target engagement by p217 + tau-targeting immunotherapeutics. CONCLUSION: The assay for measuring p217 + tau described here is highly sensitive, accurate, precise, dilution linear, and shows good potential for identifying and staging AD.


Asunto(s)
Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Proteínas tau/análisis , Proteínas tau/metabolismo , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquídeo , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados
13.
BMC Mol Cell Biol ; 21(1): 81, 2020 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-33183222

RESUMEN

BACKGROUND: Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. METHODS: Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. RESULTS: We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. CONCLUSIONS: This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Agregación Patológica de Proteínas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Epítopos , Células HEK293 , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Unión Proteica , Proteínas Recombinantes , Sonicación , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas tau/genética , Proteínas tau/ultraestructura
14.
J Alzheimers Dis ; 77(4): 1397-1416, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32894244

RESUMEN

BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.


Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales/metabolismo , Descubrimiento de Drogas/métodos , Proteínas tau/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas tau/química
15.
Mol Pharmacol ; 75(3): 648-57, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19098121

RESUMEN

The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF(1) and CRF(2(a)) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of Galpha(q/11) and Galpha(16) led to leftward shifts of the agonist concentration-response curves. Blockade of Galpha(q/11) proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF(1)- and CRF(2(a))-expressing HEK293 cells, thereby proving a contribution of the G(q) protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G(i/o) proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gbetagamma subunits to phospholipase C. G(i/o) inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF(1) and CRF(2(a)) receptors, thereby demonstrating natural coupling of G(i) proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G(s) signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G(s) and G(q) on the calcium signaling and, hence, that they act in parallel. On the other hand, G(i) coupling required prior G(s) activation.


Asunto(s)
Señalización del Calcio/fisiología , Receptores de Hormona Liberadora de Corticotropina/fisiología , Hormona Adrenocorticotrópica/análogos & derivados , Hormona Adrenocorticotrópica/fisiología , Línea Celular , AMP Cíclico/fisiología , Humanos , Receptores de Hormona Liberadora de Corticotropina/agonistas , Transfección
16.
Mol Endocrinol ; 22(6): 1464-75, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18337590

RESUMEN

Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.


Asunto(s)
Tejido Adiposo/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ghrelina/farmacología , Receptores Acoplados a Proteínas G/fisiología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Femenino , Tracto Gastrointestinal/metabolismo , Genes fos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos
17.
J Neurochem ; 104(1): 1-13, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17971128

RESUMEN

Despite the apparent homology in the protein kinase C (PKC) family, it has become clear that slight structural differences are sufficient to have unique signalling properties for each individual isoform. For PKCepsilon in depth investigation of these aspects revealed unique actions in the CNS and lead to development of specific modulators with clinical perspective. In this review, we describe to which extent PKCepsilon is distinct from other isoforms on the level of tissue expression and protein structure. As this kinase is highly expressed in the brain, we outline three main aspects of PKCepsilon signalling in the CNS. First, its ability to alter the permeability of N-type Ca2+ channels in dorsal root ganglia has been shown to enhance nociception. Secondly, PKCepsilon increases anxiety by diminishing GABA(A)R-induced inhibitory post-synaptic currents in the prefrontal cortex. Another important aspect of the latter inhibition is the reduced sensitivity of GABA(A) receptors to ethanol, a mechanism potentially contributing to abuse. A third signalling cascade improves cognitive functions by facilitating cholinergic signalling in the hippocampus. Collectively, these findings point to a physical and behavioural sensitising role for this kinase.


Asunto(s)
Sistema Nervioso Central/metabolismo , Nociceptores/fisiología , Proteína Quinasa C-epsilon/fisiología , Transducción de Señal/fisiología , Animales , Conducta Animal/efectos de los fármacos , Cognición/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Modelos Biológicos , Proteína Quinasa C-epsilon/química
18.
Acta Neuropathol Commun ; 6(1): 59, 2018 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-30001207

RESUMEN

Aggregation of tau protein and spreading of tau aggregates are pivotal pathological processes in a range of neurological disorders. Accumulating evidence suggests that immunotherapy targeting tau may be a viable therapeutic strategy. We have previously described the isolation of antibody CBTAU-22.1 from the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to specifically bind a disease-associated phosphorylated epitope in the C-terminus of tau (Ser422) and to be able to inhibit the spreading of pathological tau aggregates from P301S spinal cord lysates in vitro, albeit with limited potency. Using a combination of rational design and random mutagenesis we have derived a variant antibody with improved affinity while maintaining the specificity of the parental antibody. This affinity improved antibody showed greatly enhanced potency in a cell-based immunodepletion assay using paired helical filaments (PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the affinity improved antibody limits the in vitro aggregation propensity of full length tau species specifically phosphorylated at position 422 produced by employing a native chemical ligation approach. Together, these results indicate that in addition to being able to inhibit the spreading of pathological tau aggregates, the matured antibody can potentially also interfere with the nucleation of tau which is believed to be the first step of the pathogenic process. Finally, the functionality in a P301L transgenic mice co-injection model highlights the therapeutic potential of human antibody dmCBTAU-22.1.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Anticuerpos/farmacología , Encéfalo/metabolismo , Serina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Afinidad de Anticuerpos/efectos de los fármacos , Autopsia , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Epítopos/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Mutagénesis , Mutación/genética , Fosforilación/fisiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/terapia
19.
J Alzheimers Dis ; 65(1): 265-281, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30040731

RESUMEN

The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.


Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/metabolismo , Encéfalo/metabolismo , Proteínas tau/inmunología , Animales , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Resonancia por Plasmón de Superficie , Proteínas tau/deficiencia , Proteínas tau/genética
20.
Acta Neuropathol Commun ; 6(1): 43, 2018 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-29855358

RESUMEN

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.


Asunto(s)
Linfocitos B/metabolismo , Inmunoglobulina G/farmacología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Proteínas tau/inmunología , Proteínas tau/metabolismo , Adolescente , Adulto , Anciano , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Cristalización , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Epítopos Inmunodominantes/metabolismo , Masculino , Microglía/metabolismo , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Agregado de Proteínas , Adulto Joven
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